PROJECT ABSTRACT Neural signals generated by inner hair cells (IHCs) are transmitted to higher brain center by a functionally heterogeneous population of Type I spiral ganglion neurons (SGNs) in the inner ear. Although Type I SGNs have been grouped into three physiological classes based on basal firing rates, in the absence of molecular correlates, it has been difficult to study their development or the basis of their differential vulnerability to acoustic overexposure. We recently conducted single cell RNA-sequencing (scRNA-seq) and uncovered three broad Type I SGN molecular subtypes that exhibit the same distinctions in peripheral anatomy and synaptic features found among physiologically defined subgroups. In addition, we found that refinement of molecular segregation, which is apparent shortly after birth, depends on spontaneous activity in the first postnatal week. Some evidence, however, support a model in which the earliest molecular segregation of Type I SGNs occurs embryonically, perhaps independent of activity. Here we propose to study the early developmental appearance of SGN identities and their malleability in adulthood using a transcriptome-based approach. In Aim 1, we investigate emergence of molecular heterogeneity at embryonic stages and test the requirement of IHCs for this process. In Aim 2, we determine the temporal window over which Type I SGNs undergo changes in molecular identity upon loss of IHC-driven excitation and seek to identify a transcription factor code that can mediate switching of subtype molecular identity in mature SGNs. Together, we anticipate that completion of these aims will facilitate hypothesis-driven inquiries into the mechanisms underlying early segregation of SGN identities, and inform approaches to manipulate the molecular profiles of SGNs in adulthood. Our long-term goal is to gain a mechanistic understanding of how extrinsic effectors influence cell-intrinsic programs to generate distinct SGN identities during development and utilize that conceptual framework to alter gene expression states in mature neurons toward therapeutic end goals.